JPH0551321A - Immune activating agent - Google Patents

Abstract

PURPOSE:To provide an immune activating agent containing cell bodies of a specified bacterium belonging to Bifidobacterium genus as the active component and effective especially for therapy of gastrointestinal diseases, infectious diseases, etc. CONSTITUTION:One or more kinds of cell bodies selected from Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium infantis and Bifidobacterium adolescentis are contained as the active component. The above-mentioned microorganism is used in a form of living cells, died cells killed by heat treatment, etc., freeze-dried cell bodies, etc. The above-mentioned microorganism is remarkably excellent in improvement of immunological response of the lymph node cells, low in activation effect for the spleen cells, lacking in activation effect for the thymocyte and effective for improving the activity of macrophage by synergism with a very small amount of lipopolysaccharide not showing a remarkable effect by itself.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunostimulant, and more particularly to an immunostimulant containing, as an active ingredient, microbial cells of the genus Bifidobacterium which is an intestinal bacterium.

[0002]

2. Description of the Related Art Bifidobacteriu
m) genus bacterium is a kind of intestinal bacterium, and is known to be effective against digestive diseases and infectious diseases by improving the intestinal flora to the predominance of Bifidobacterium. .. In particular, it has been reported that one of the causes that breast-fed infants are less susceptible to dyspepsia and infectious diseases than artificially-fed infants is that the proportion of Bifidobacterium bacteria in the intestinal flora is high. .. On the other hand, with regard to the immunological action of Bifidobacterium, Bifidobacterium infantis (B. infantis) non-specifically activates B cells and macrophages of lymphocytes, and also causes syngeneic transplantation. Healing the intestine (kohwi, Y. et al: Gann, 69: 613, 1978)
But moreover, Bifidobacterium breve (B.bre
ave) has the effect of enhancing the antibody production of Peyer's patch cells and promoting the proliferation of bone marrow-derived B cells (Hosai et al .;
JP-A-1-242532) has been reported.

[0003]

However, there are very few examples of systematic and detailed examination of the difference in immunological properties between Bifidobacterium and other edible bacteria so far. An object of the present invention is to clarify the degree of immunostimulatory action of Bifidobacterium, and further to analyze the action mechanism on the immune system from the action on macrophages.

[0004]

The present invention relates to at least one of Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium infantis and Bifidobacterium adrensetis. The present invention provides an immunostimulant characterized by containing the bacterial cell of (1) as an active ingredient.

The above-mentioned microorganisms used in the present invention are not particularly limited, and are stored in institutions such as ATCC and IFO and can be obtained from publicly known bacteria listed in the catalog or from Japan Bifidobacteria Center, etc., and commercially available. Any strain such as those described above can be used.
In addition to living cells, these microorganisms are also used in forms such as those killed by heat treatment or freeze-dried cells.
If desired, they may be formulated into powders, tablets, drinks and the like.

In order to solve the above-mentioned problems of the present invention, the present inventors used four kinds of test methods in order to compare the degree of immunostimulatory action of Bifidobacterium and other edible bacteria. That is, a T cell proliferation test using a mouse previously immunized with a protein antigen irrelevant to bacterial cells, B
An antibody production test by cells and a lymphocyte blast transformation test using non-immunized mice were performed. In addition, in order to analyze the action mechanism of Bifidobacterium bacteria on the immune system, the action on macrophages was also examined.

[0007]

As described above, as a result of comparing the immunostimulatory action of edible bacteria including Bifidobacterium using four types of test methods, it was found that Bifidobacterium showed a very high immune response to lymph node cells. It was found that the activity of spleen cells was increased, but that there was no active effect on thymocytes. From the above, it is clear that Bifidobacterium bacteria have an action of nonspecifically activating the immune system of the host as compared with other edible bacteria. Furthermore, since the activating effect on spleen cells was weak and the activating effect on thymocytes was not shown, the effect of Bifidobacterium bacteria on macrophages was examined. As a result, it was revealed that the Bifidobacterium bacterium has an action of enhancing the activity of macrophages by coacting with a minute amount of lipopolysaccharide (LPS) which is not very active in itself.

[0008]

The details of the present invention will now be described with reference to the following examples. The strains and culture conditions used in the experiment are shown below.

[0009]

[Table 1]

Each strain was cultivated using a frozen or freeze-dried product, and then centrifuged (10,000 rpm, 10 minutes) to recover the bacterial cells, which were resuspended in distilled water and then subjected to the same conditions. It was collected by centrifugation. Then, the collected cells were lyophilized and then sonicated at a concentration of 1 mg / ml for 15 minutes.

Example 1 Comparison of immunostimulatory action of edible bacteria containing Bifidobacterium genus Lymph node cell proliferation test Complete adjuvant (Mycobocterium tuberculo)
(including isis H37Ra strain) together with emulsified chicken egg ovomucoid (50 μg / head) on both foot pads of the mouse.
And immunize the base of tail (200 μl / head),
Two weeks later, the lymph node cells were taken out to prepare a single cell suspension. RPMI containing 1% normal mouse serum
1640 medium (100U / ml crystalline penicillin G potassium, 100μ
g / ml streptomycin sulfate, containing 5 × 10 ⁻⁵ M 2-mercaptoethanol) together with each bacterium 3.
The cells were cultured in ^{5 to} 4.0 × 10 5/200 μl / well (96 well flat bottom plate). The culture was performed under the conditions of 5% CO ₂ and 37 ° C. (Hereafter, cell culture was performed under these conditions.) After 3 days,
³ H-thymidine was added at 1 μCi / well, the culture was terminated after 18 to 22 hours, and the amount of ³ H-thymidine incorporated was measured by a liquid scintillation counter.

As a result, as shown in FIG. 1, when Bifidobacterium was added to the medium, all five strains promoted the proliferation of lymph node cells against the heterologous antigen.

Production test of anti-ovomucoid antibody by B cells In the same manner as the above-mentioned method, chicken egg ovomucoid was previously immunized, and its lymph node cells were taken out at 2 × 10 ⁶ cells.
The cells were cultured together with each of the cells at a concentration of cells / ml / well (48 well flat bottom plate). Half of the culture medium was exchanged on the 3rd and 7th days after the start of the culture, and the production amount of anti-ovomucoid antibody in the culture supernatant on the 11th day was measured by the ELISA method. As a result, as shown in FIG. 2, in this test as well, all 5 strains of Bifidobacterium increased the production of anti-ovomucoid antibody.

Lymphocyte Juvenile Test Lymphocytes were prepared from the spleen or thymus of non-immunized mice, and were used together with each of the cells using RPMI 1640 medium containing 1% normal mouse serum to obtain 5.0 × 10 5. ⁵ / 200μl / well
It was seeded on a 96-well plate at the concentration of 2 and cultured for 2 days. Thereafter, the amount of ³ H-thymidine incorporation was measured in the same manner as in the above method. The results are shown in Fig. 3. As is clear from the figure, when spleen cells were used, slightly higher activity was observed for all Bifidobacterium bacteria, but when thymocytes were used, no activity was observed for any strain. There wasn't. This suggests that these strains do not act directly on thymic T cells. From the above results, it was confirmed that Bifidobacterium bacteria nonspecifically activate the immune system and have an immunostimulatory action specific to the genus.

Example 2 Effect of Bifidobacterium bacteria on macrophages Ce of macrophage by Bifidobacterium bacteria
The activating action of the RAW 264.7 strain, which is an ll line, is one of the lymphocyte activating factors, interleukin-1 (hereinafter,
Abbreviated as IL-1. ) Was used as an index.

48 wells containing macrophages together with each bacterial cell
Spread on a flat-bottom plate at a concentration of 5 × 10 ⁵ cells / well / ml, 24
After culturing for a time, the culture supernatant was collected. Also, a test was carried out by adding a small amount of LPS (positive control for macrophage activation), which has low activity per se, to the bacterial cells. The concentration of IL-1 contained in the supernatant was D10G, which is a cell line of IL-1 dependent cells and helper T cells.
It was calculated by the degree of proliferation in 4.1.

As shown in FIG. 4, the Bifidobacterium bacterium did not promote the production of IL-1 by either of the two strains, but the addition of a trace amount of LPS significantly increased the amount of I-1.
It stimulated the production of L-1. That is, it was revealed that the Bifidobacterium bacterium cooperates with a small amount of LPS to promote the activation of macrophages.

[0018]

INDUSTRIAL APPLICABILITY As is apparent from the above description, the present invention takes advantage of the fact that a specific Bifidobacterium bacterium has an apparently higher immunostimulatory action than other edible bacteria. The drug is provided, and is particularly effective in treating diseases of the digestive system and infectious diseases. Further, the Bifidobacterium bacterium used in the present invention is a microorganism that is a useful bacterium in the intestine and is widely used in yogurt and the like, and thus is safe to be taken orally and applied to a wide range of foods. it can.

[0019]

[Brief description of drawings]

FIG. 1 shows the results of lymph node cell proliferation tests of edible bacteria including Bifidobacterium.

FIG. 2 shows the results of an anti-ovomucoid antibody production test by B cells of lymph node cells of edible bacteria including Bifidobacterium.

FIG. 3 shows the results of a lymphocyte blast transformation test of edible bacteria including Bifidobacterium.

FIG. 4 shows the results of tests of edible bacteria including Bifidobacterium bacteria on macrophages.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Etsuo Minagawa 465-1 Waatsu, Hiroshima-cho, Sapporo-gun, Hokkaido Inside Yotsuba Dairy Co., Ltd. Research Center (72) Fuji-shi Shiji 465-1 Wa-atsu, Hiroshima-cho, Sapporo-gun, Hokkaido Address Yotsuba Dairy Co., Ltd. Research Center (72) Inventor Shuichi Uenogawa 400-8 Masuda Nitta Kasukabe City, Saitama Prefecture

Claims (1)

[Claims] 1. At least one bacterial cell selected from the group consisting of Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium infantis and Bifidobacterium adrensetis as an active ingredient. An immunostimulant characterized by comprising: